The Major Intrinsic Protein (MIP) family is large and diverse, possessing over 100 members that all form transmembrane channels. MIPs facilitate the passive transport of small polar molecules across membranes. These channel proteins function in water, small carbohydrate (e.g., glycerol), urea, NH3, CO2 and possibly ion transport by an energy independent mechanism. MIPs constitute a very old family of proteins and are found ubiquitously in all kinds of living organisms, including bacteria, fungi, animals, and plants. Phylogenetic clustering of the proteins is largely according to phylum of the organisms of origin, but one to three clusters are observed for each phylogenetic kingdom (plants, animals, yeast, bacteria and archaea). One of the plant clusters includes only tonoplast (TIP) proteins, with another includes plasma membrane (PIP) proteins (for a review see Urban Johanson (2001) Plant Physiol. 126(4): 1358-1369).
In the genomic sequence of Arabidopsis, 35 different MIP-encoding genes were identified. Based on sequence similarity, these 35 proteins are divided into four different sub-families: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The Arabidopsis TIP proteins are encoded by the genes described by the following locii: At2g36830 (TIP1;1 tonoplast intrinsic protein gamma 1), At1g73190 (TIP1;2 tonoplast intrinsic protein alpha), At4g01470 (TIP1;3 tonoplast intrinsic protein gamma 3), At3g16240 (TIP2;1 tonoplast intrinsic protein delta 1), At4g17340 (TIP2;2 tonoplast intrinsic protein delta 2), At5g47450 (TIP2;3 tonoplast intrinsic protein delta 3), At1g73190 (TIP3;1 tonoplast intrinsic protein alpha), At1g17810 (TIP3;2 tonoplast intrinsic protein beta), At2g25810 (TIP4;1 tonoplast intrinsic protein epsilon), At3g47440 (TIP5;1 tonoplast intrinsic protein zeta). For nomenclature of the various TIP proteins see Urban Johanson (2001) Plant Physiol. 126(4): 1358-1369.
With few exceptions, a strict organ-specific expression has not been found for Arabidopsis MIP genes. However, preferential expression in seeds/embryos, roots, and shoots has been found for some TIP genes (profiles compared in Urban Johanson (2001) Plant Physiol. 126(4): 1358-1369). AtTIP3;1 (TIP alpha) are described to be seed- and embryo-specific AQP in Arabidopsis and other plants such as Phaseolus vulgaris and Ricinus communis (Johnson K D et al. (1989) Plant Physiol 91:1006-1013; van de Loo F J et al. (1995) Plant Physiol 108:1141-1150). AtTIP1;1 is expressed mainly in the elongation zone of roots and to lower levels in various shoot organs (Höfte H et al. (1992) Plant Physiol 99:561-570; Ludevid D et al. (1992) Plant Physiol 100:1633-1639). It is interesting that this elongation-associated AQP can be induced by gibberellic acid, which is known to promote cell growth in Arabidopsis dwarf mutants (Phillips A L et al. (1994) Plant Mol Biol 24:603-615). In contrast, AtTIP2;1 is mainly expressed in shoots and to a lower extent in roots (Daniels M J et al. (1996) Plant Cell 8:587-599). No expression profile is so far reported for the AtTIP1;2 gene. Based on the above described heterogeneity of the expression profiles a prediction of the specificity for that gene does not seem to be possible.
It is however very difficult to distinguish between the certain TIP homologous proteins (e.g., between alpha and beta) and therefore difficult to predict the expression profile for a TIP gene. For example the TIP-alpha und beta genes are very similar on protein level (see FIG. 1) but characterized by distinct expression profiles. While the so-called TIP-alpha proteins are more expressed in seed the TIP-beta proteins are expressed (for Arabidopsis) also in rosette leafs and etiolated seedlings (Naoto Mitsuhashi et al. The Plant Cell, Vol. 13, 2361-2372, October 2001; Jiang L, Rogers J C., J Cell Biol. 1998 Nov. 30; 143(5):1183-99; Quigley F et al. Genome Biology 2001, 3(1):research0001.1-0001.17).
Furthermore, it is not guaranteed that the expression profile of an endogenous promoter for its endogenous gene can be easily utilized for transgenic expression techniques. Often essential elements are either in introns or in trans-regions, which are removed during the isolation of the promoter region, thereby significantly changing the expression profile of the promoter.
Manipulation of plants to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues. Such genetic manipulation relies on the availability of a means to drive and to control gene expression as required. For example, genetic manipulation relies on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. For numerous applications in plant biotechnology a tissue-specific expression profile is advantageous, since beneficial effects of expression in one tissue may have disadvantages in others. Seed-preferential or seed-specific promoters are useful for expressing genes as well as for producing large quantities of protein, for expressing oils or proteins of interest, e.g., antibodies, genes for increasing the nutritional value of the seed and the like. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. Moreover, the increasing interest in cotransforming plants with multiple plant transcription units (PTU) and the potential problems associated with using common regulatory sequences for these purposes merit having a variety of promoter sequences available.
There is, therefore, a great need in the art for the identification of novel sequences that can be used for expression of selected transgenes in economically important plants. It is thus an objective of the present invention to provide new and alternative expression cassettes for seed-preferential or seed-specific expression of transgenes in plants. The objective is solved by the present invention.